Dendritic cells (DCs) are sentinels of the peripheral immune system that continuously survey their surroundings for pathogens, by fluid phase uptake and endocytosis. When pathogen-derived fragments are detected, DCs engulf the foreign material and in the process become activated. Engulfed proteins are processed and displayed as peptide/MHC complexes to Class II MHC-restricted T cells, while lipids are incorporated into CD1/(32m/lipid complexes for presentation to CD1-restricted T cells. Much of what is known about endosomal antigen processing, loading and presentation of Class II MHC molecules was learned through biochemical and cell biological analysis of B cell lines, and to a more limited extent, of cultured DCs. New methodology now makes it possible to visualize the uptake of red fluorescent antigen by live Class II MHC-positive DCs in situ in the epidermis of mice that express green fluorescent protein (GFP)-Class II MHC fusion proteins. Trafficking of antigen-experienced DCs to skin-draining lymph nodes can be tracked in live cells. First, we will study the protein and lipid antigen-presentation pathways in skin-derived DCs and lymph node-resident DCs. We will test our hypothesis that during inflammation, priming of immune responses in the skin requires cell-mediated transport of both protein and lipid antigens by epidermis- resident DCs (Langerhans cells) to skin-draining lymph nodes. Antigen presentation by DCs that reside in the T cell area of lymph nodes is essential for T cell priming. We hypothesize that antigen transfer from LC to a non-migratory lymph node-resident DCs is necessary for successful T cell priming to occur. Accordingly, mutant mice in which LCs express normal antigen endocytosis but defective endosomal processing should have limited phenotype. In contrast, mice expressing non-migratory DCs defective in endosomal processing should exhibit reduced T cell priming capacities. We will study antigen processing and Class II MHC presentation pathways in epidermis-derived Langerhans cells and lymph node-resident DC subtypes. Finally, we have succeeded to generate a new mouse model to study CD1-mediated antigen presentation in vivo. The possible involvement of CD1d to immune responses originating in the skin will be investigated in CD1d- yellow fluorescent protein knock-in mice.